Our overall objective is to characterize experimentally the mechanism of folding in small proteins, especially ribonuclease A (RNase A), by detecting and characterizing kinetic intermediates in the reversible unfolding/refolding process. A study of folding transition of trypsin inhibitor (BPTI) is also being started. We have found that there are two forms of unfolded RNase A, a fast-refolding and a slow-refolding form, and one specific aim is to find out the nature of the difference between these two forms and also to determine whether other small proteins also show both fast-refolding and slow-refolding forms. Recently we have found a structural intermediate that is found at an early stage in the folding both of RNase A and of the modified species RNase S, which dissociates into the 20-residue S-peptide and 104-residue S-protein. Creation of a functional binding site for S-peptide is part of this early folding process. A second specific aim is to characterize further the structure present in this early folding intermediate, especially by kinetic NMR studies of folding at low temperatures. Another kinetic intermediate has been found at an early stage in the unfolding of RNase A at pH 3.0, and a third specific aim is to characterize this intermediate.